A new cell line was created using CHO-GS system. The most promising clones were adapted to different base cultivation media leading to the selection of one medium. The fed-batch process development was performed in spinner, shake flask and bioreactor scale. It included the selection of a feed medium, the choice of the feed strategy and the optimisation of the glucose feeding. The process was then simplified by using a single feed including the feed medium and the glucose feed. Finally up-scaling parameters like aeration and CO2 stripping were studied in 3 L and 15 L bioreactors in preparation for pilot scale operation. This process proved to be robust, reproducible and suitable for large and commercial scale operation.

This work aimed at studying the impact of alkali addition in a Chinese Hamster Ovary cell culture. Two phenomena were studied, the kinetic rate of direct cell death in presence of high pH and the effect of transitory single contact of high pH on cell viability and growth. Contact with pH 11 or 10 did not provoke immediate cell lysis. The cells survived several minutes to such conditions. Contact with pH 11 during 2 minutes, with pH 10 during 5 minutes, with pH 9 during 5 minutes or 10 minutes did not affect the viability. In these conditions, the growth was not affected except after 5 minutes contact at pH 10 or 10 minutes contact at pH 9 for which the growth was slowed down the first day only. As expected, NaOH addition affected the cells more than Na2CO3 addition. This was due to a higher pH but could be even observed at the same pH (10).

A process for cultivating animal cells producing complex proteins, wherein one plant-derived peptone or a combination of plant-derived peptones is fed to the cell culture, as well as a method for reducing the toxic effect of over-feeding amino acids during a fed-batch process for cultivating animal cells producing complex proteins.

Eight commercial peptones, derived from plants, were studied for their ability of improving the cell growth and the productivity of a CHO cell line producing a human monoclonal antibody. They were also compared to yeast, lactalbumin and meat derived peptones. Seven plant peptones were selected and further studied in combination by Design of Experiment. The best three peptones were then tested in combinations in fed-batch cultivation. The fed-batch process was based on low concentrations of glucose and glutamine with feeding of amino acids, peptones and feed medium including vitamins, metal traces and biosynthesis precursors. This process was based on Biovitrum protein-free proprietary medium for the base medium and the feeding medium. Different feeding strategies, different peptone combinations and phosphate feeding were studied for their ability to improve the cell density, the cell specific productivity and the cultivation longevity

Major advantages of perfusion are high cell numbers and high total production in a relatively small size bioreactor. Moreover, perfusion is optimal when the product of interest is unstable or if the product yield is low. On the other hand, disadvantages are for example technical challenges originating from non-robust cell separation devices as well as sterility concerns from the more complex set-up needed.

In the present work, the use of a WAVE Bioreactor™ system 20/50 in perfusion mode with10 L disposable Cellbag™ bioreactors customized with two dip tubes in combination with disposable hollow fiber filters as external cell separating devices were investigated. A comparison between Alternating Tangential Flow (ATF) and Tangential Flow Filtration (TFF) was performed using a recombinant CHO cell line producing a monoclonal antibody (mAb) as a model system.

This project aimed to find a method for producing potential probe targets for identification of ESBL (Extended Spectrum Beta Lactamase) genes in bacteria. ESBLs are a type of enzymes responsible for antibiotic resistance in many bacteria. The result we developed was a semi-automated pipeline that utilises several Perl scripts to download gene sequences, identify sequence subgroups based on sequence similarity, find common target sequences among them and screen the target sequences against a background database. These target sequences should work with padlock probes and therefore had specific requirements regarding length and highest number of allowed mismatches. This report includes descriptions of the scripts and ideas for future improvements, as well as an ethical analysis about aspects relevant to research on antibiotic resistance.

Salmonella Typhimurium causes a self-limiting gastroenteritis that may lead to systemic disease. Bacteria invade the small intestine, crossing the intestinal epithelium from where they are transported to the mesenteric lymph nodes (MLNs) within migrating immune cells. MLNs are an important site at which the innate and adaptive immune responses converge but their architecture and function is severely disrupted during S. Typhimurium infection. To further understand host-pathogen interactions at this site, we used mass spectrometry imaging (MSI) to analyse MLN tissue from a murine model of S. Typhimurium infection. A molecule, identified as palmitoylcarnitine (PalC), was of particular interest due to its high abundance at loci of S. Typhimurium infection and MLN disruption. High levels of PalC localised to sites within the MLNs where B and T cells were absent and where the perimeter of CD169(+) sub capsular sinus macrophages was disrupted. MLN cells cultured ex vivo and treated with PalC had reduced CD4(+) CD25(+) T cells and an increased number of B220(+) CD19(+) B cells. The reduction in CD4(+) CD25(+) T cells was likely due to apoptosis driven by increased caspase-3/7 activity. These data indicate that PalC significantly alters the host response in the MLNs, acting as a decisive factor in infection outcome.

A serum-free fed-batch process was developed for production of a human monoclonal antibody in Chinese hamster ovary (CHO) cells based on Biovitrum’s proprietary low protein serum-free medium without animal derived components (BVT4). The cells were fed with glucose, glutamine and Biovitrum’s proprietary low protein serum-free feed medium without animal derived components enriched with amino acids, vitamins, metal traces, peptones, and biosynthesis precursors. To improve the performance of the fed-batch process, we developed the use of plant peptones by studying the dose and timing of the peptone feeding. Different doses of peptone cocktail and amino acid cocktail, as well as different combinations of pep- tone and amino acid cocktails were first screened in 50 ml filter tubes on an AgCell shaker table. The best combinations were then assessed in spinner and 3 L bioreactor cultures. To reinforce our findings, the antibody-producing CHO cells were adapted to a disclosed serum-free medium DMEM/F12 and the beneficial effects of pep- tones were confirmed in a fed-batch process based on the DMEM/F12 serum-free medium.

To date there is no accepted clinical diagnostic test for Parkinson's disease (PD) based on biochemical analyses of blood or cerebrospinal uid. Currently, diagnosis, measurement of disease progression and response to therapeutic intervention are based on clinical observation, but the rst neuronal dysfunction precede the earliest recognition of symptom by at least 5 - 10 years. A potential diagnostic biomarker is oligomeric alpha-synuclein which in recent papers have reported a signicant quantitative dierence between PD and controls. In this master thesis, a method for measuring oligomeric levels of alpha-synuclein is presented together with a monomeric measuring commercial kit used to measure alpha-synuclein in a preclinical model of PD. A signicant dierence of monomeric levels could be detected between two weeks and four weeks post injection of a vector containing the gene for human alpha-synuclein, no signicant dierence between four and eight weeks was found.

The pathogenic bacterium Mycobacterium tuberculosis (Mt) is the causative agent of tuberculosis, a widespread and fatal infectious disease. Today, treatment against tuberculosis involves a combination of drugs, which need to be taken for at least six months and which often causes severe side effects. Therefore, new drugs that are more effective and that give fewer side effects are needed. A characteristic feature of the Mt bacterium is its very complex and thick cell wall, which prevents many potential drug molecules from penetrating it. Inhibiting any one of the enzymes that are involved in its biosynthesis would therefore seem to be a good strategy for eliminating the Mt bacteria. The aim of this study was to characterize four enzymes involved in Mt cell wall biosynthesis. In order to do that, they were produced recombinantly in E. coli and purified. Crystallization experiments were set up in order to produce diffracting crystals, with the aim of structure determination and drug design.

On the road to success, companies need to fulfil their stakeholders’ expectations. On the road to business excellence, companies need to exceed these expectations. Oliver Wight Inc. has established a certification called Class A Business, which shows that a company is exceeding stakeholders’ expectations and that it performs in the upper quartile in its respective industry. The Class A Business certification is awarded, once a company fulfils a certification checklist with Class A Business criteria. To get to this point, a company can design their road to business excellence by following a specific set of nine Class A Behaviors.

This study focuses on four of these behaviors, divided in three parts, and how they are implemented at a pharmaceutical packaging department at Roche in Kaiseraugst, Switzerland. In addition, through employee feedback potential areas of improvement are identified.

For a company to understand how it is running, it has to understand its underlying processes. Once the processes are in place, a process-oriented way of thinking can change a company to make decisions based on process’ needs rather than on individual preferences. Business processes and their potential for continuous improvement were the first part of the study. The second part of the study investigated the communication of different functions in the packaging process and how the flow of information could be improved. In the third part, the usage of operational metrics in the packaging department is researched by a user feedback survey.

An innovative way to visualize meeting conversations was developed in this study to make meetings more tangible for the reader. This is a newly developed and never before described method for business research colorfully showing interactions in meetings.

The results are very intriguing. Simple thought business elements seem to pose larger hurdles than would be expected sometimes.

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly used classes of drugs. They target the cyclooxygenases (COX) 1 and 2 to reduce the physiological responses of pain, fever, and inflammation. Due to their role in inducing angiogenesis, COX proteins have also been identified as targets in cancer therapies.

In this thesis, I describe computational protocols of molecular docking, molecular dynamics simulations and free energy calculations. These methods were used in this thesis to determine structure-activity relationships of a diverse set of NSAIDs in binding to their target proteins COX-1 and 2. Binding affinities were calculated and used to predict the binding modes. Based on combinations of molecular dynamics simulations and free energy calculations, binding mechanisms of sub-classes of NSAIDs were also proposed. Two stable conformations of COX were probed to understand how they affect inhibitor affinities. Finally, a brief discussion on selectivity towards either COX isoform is discussed. These results will be useful in future de novo design and testing of third-generation NSAIDs.

The percolation theory is established as a useful tool in the field of pharmaceutical materials science.It is shown that percolation theory, developed for analyzing insulator–conductor transitions, can beapplied to describe imperfect dc conduction in pharmaceutical microcrystalline cellulose duringdensification. The system, in fact, exactly reproduces the values of the percolation threshold andexponent estimated for a three-dimensional random continuum. Our data clearly show a crossoverfrom a power-law percolation theory region to a linear effective medium theory region at a celluloseporosity of ;0.7.

Our increasing understanding of disease mechanisms coupled with technological advances has facilitated the generation of pharmaceutical proteins, which are able to address yet unmet medical needs. Diseases that were fatal in the past can now be treated with novel biological medications improving and prolonging life for many patients. Pharmaceutical protein production is, however, a complex undertaking, which is by no means problem-free. The demand for more complex proteins and the realization of the importance of post-translational modifications have led to an increasing use of mammalian cells for protein expression. Despite improvements in design and production, the costs required for the development of pharmaceutical proteins still are far greater than those for conventional, small molecule drugs. To render such treatments affordable for healthcare suppliers and assist in the implementation of precision medicine, further progress is needed. In five papers this thesis describes strategies and methods that can help to advance the development and manufacturing of pharmaceutical proteins. Two platforms for antibody engineering have been developed and evaluated, one of which allows for efficient screening of antibody libraries whilst the second enables the straightforward generation of bispecific antibodies. Moreover, a method for epitope mapping has been devised and applied to map the therapeutic antibody eculizumab’s epitope on its target protein. In a second step it was shown how this epitope information can be used to stratify patients and, thus, contribute to the realization of precision medicine. The fourth project focuses on the cell line development process during pharmaceutical protein production. A platform is described combining split-GFP and fluorescence-activated droplet sorting, which allows for the efficient selection of highly secreting cells from a heterogeneous cell pool. In an accompanying study, the split-GFP probe was improved to enable shorter assay times and increased sensitivity, desirable characteristics for high-throughput screening of cell pools. In summary, this thesis provides tools to improve design, development and production of future pharmaceutical proteins and as a result, it makes a contribution to the goal of implementing precision medicine through the generation of more cost-effective biopharmaceuticals for well-characterized patient groups.

Matters of the HAART! The current treatment for human immunodeficiency virus (HIV), HAART, makes use of a combination of antiretroviral drugs, which are poorly soluble in aqueous media. Enhancing the solubility of such drugs through the use of mesoporous materials could lead to improved treatment efficiency and might provide a solution to the drug-drug interaction problems associated with these types of therapeutic regimes.